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Background: Layilin (LAYN) represents a valuable prognostic biomarker across various tumor types, while also serving as an innovative indicator of dysfunctional or exhausted CD8+ T cells and exhibiting correlation with immune context. However, the immune function and prognostic significance of LAYN in hepatocellular carcinoma (HCC) remain unexplored. Therefore, our objective is to investigate the role of LAYN in CD8+ T cell exhaustion, clinical prognosis, and the tumor microenvironment within HCC. Methods: TIMER or GEPIA databases were used to analyze LAYN expression level and its correlation with immune infiltration in HCC. Bioinformatics analysis was conducted on TCGA and scRNA-seq cohorts. The evaluation of LAYN expression level in fresh specimens was performed through IF, IHC, and ELISA assays. Flow cytometry and mRNA-seq were employed to investigate co-expressed genes of LAYN, the LAYN+CD8+ T cell exhaustion signature and immune function. Cell proliferation ability and killing activity were assessed using CCK8 and CFSE/PI. Results: The expression level of LAYN in HCC tumors was significantly higher compared to peri-tumors. Patients with high levels of LAYN exhibited poorer OS. GO or KEGG analysis confirmed that LAYN was involved in immune response and was positively associated with CD8+ T cell immune infiltration levels. Furthermore, LAYN negatively regulated the immune function of CD8+ T cells, leading to dysfunctional phenotypes characterized by elevated levels of CD39, TIM3 and reduced levels of perforin, TNF-α, Ki-67. CFSE/PI assays demonstrated that LAYN+CD8+ T cells displayed decreased cytotoxic activity. Additionally, there was a positive correlation between LAYN and CD146 levels, which are involved in adhesion and localization processes of CD8+ T cells. Interestingly, blocking LAYN partially restored the exhaustion properties of CD8+ T cells. Conclusion: LAYN exhibits a strong correlation with immune infiltration in the TME and represents a novel biomarker for predicting clinical prognosis in HCC. Moreover, targeting LAYN may hold promise as an effective strategy for HCC immunotherapy.
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Further reducing total nitrogen (TN) and total phosphorus (TP) in the secondary effluent needs to be realized effectively and in an eco-friendly manner. Herein, four pyrite/sawdust composite-based biofilters were established to treat simulated secondary effluent for 304 days. The results demonstrated that effluent TN and TP concentrations from biofilters under the optimal hydraulic retention time (HRT) of 3.5 h were stable at <2.0 and 0.1 mg/L, respectively, and no significant differences were observed between inoculated sludge sources. The pyrite/sawdust composite-based biofilters had low N2O, CH4, and CO2 emissions, and the effluent's DOM was mainly composed of five fluorescence components. Moreover, mixotrophic denitrifiers (Thiothrix) and sulfate-reducing bacteria (Desulfosporosinus) contributing to microbial nitrogen and sulfur cycles were enriched in the biofilm. Co-occurrence network analysis deciphered that Chlorobaculum and Desulfobacterales were key genera, which formed an obvious sulfur cycle process that strengthened the denitrification capacity. The higher abundances of genes encoding extracellular electron transport (EET) chains/mediators revealed that pyrite not only functioned as an electron conduit to stimulate direct interspecies electron transfer by flagella but also facilitated EET-associated enzymes for denitrification. This study comprehensively evaluates the water-gas-biofilm phases of pyrite/sawdust composite-based biofilters during a long-term study, providing an in-depth understanding of boosted electron transfer in pyrite-based mixotrophic denitrification systems.
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Biofilmes , Desnitrificação , Nitratos , Fósforo , Fósforo/metabolismo , Nitratos/metabolismo , Nitrogênio/metabolismo , Transporte de Elétrons , Ferro , SulfetosRESUMO
Metabolic reprogramming induced by Epstein-Barr virus (EBV) often mirrors metabolic changes observed in cancer cells. Accumulating evidence suggests that lytic reactivation is crucial in EBV-associated oncogenesis. The aim of this study was to explore the role of metabolite changes in EBV-associated malignancies and viral life cycle control. We first revealed that EBV (LMP1) accelerates the secretion of the oncometabolite D-2HG, and serum D-2HG level is a potential diagnostic biomarker for NPC. EBV (LMP1)-driven metabolite changes disrupts the homeostasis of global DNA methylation and demethylation, which have a significantly inhibitory effect on active DNA demethylation and 5hmC content. We found that loss of 5hmC indicates a poor prognosis for NPC patients, and that 5hmC modification is a restriction factor of EBV reactivation. We confirmed a novel EBV reactivation inhibitor, α-KG, which inhibits the expression of EBV lytic genes with CpG-containing ZREs and the latent-lytic switch by enhancing 5hmC modification. Our results demonstrate a novel mechanism of which metabolite abnormality driven by EBV controls the viral lytic reactivation through epigenetic modification. This study presents a potential strategy for blocking EBV reactivation, and provides potential targets for the diagnosis and therapy of NPC.
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Metilação de DNA , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Ativação Viral , Humanos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Carcinoma Nasofaríngeo/virologia , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/virologia , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/complicações , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/genética , Epigênese Genética , Progressão da DoençaRESUMO
Adoptive cell therapy using T cell receptor-engineered T cells (TCR-T) is a promising approach for cancer therapy with an expectation of no significant side effects. In the human body, mature T cells are armed with an incredible diversity of T cell receptors (TCRs) that theoretically react to the variety of random mutations generated by tumor cells. The outcomes, however, of current clinical trials using TCR-T cell therapies are not very successful especially involving solid tumors. The therapy still faces numerous challenges in the efficient screening of tumor-specific antigens and their cognate TCRs. In this review, we first introduce TCR structure-based antigen recognition and signaling, then describe recent advances in neoantigens and their specific TCR screening technologies, and finally summarize ongoing clinical trials of TCR-T therapies against neoantigens. More importantly, we also present the current challenges of TCR-T cell-based immunotherapies, e.g., the safety of viral vectors, the mismatch of T cell receptor, the impediment of suppressive tumor microenvironment. Finally, we highlight new insights and directions for personalized TCR-T therapy.
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Background: MicroRNA-612 (miR-612) has been proven to suppress the formation of invadopodia and inhibit hepatocellular carcinoma (HCC) metastasis by hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA)-mediated lipid reprogramming. However, its biological roles in HCC cell ferroptosis remain unclear. Methods and Results: In this study, we found that HCC cells with high metastatic potential were more resistant to ferroptosis, indicating that ferroptosis is related to HCC metastasis. The levels of lipid reactive oxygen species (ROS) were found to be much lower in HCC cells with high metastatic potential by flow cytometry (FCM). We used HCC cells with miR-612 overexpression/knockout and HADHA overexpression/knockdown to test cell viability after stimulation with RSL3. HCC cells overexpressing miR-612 were more sensitive to ferroptosis, and miR-612 could increase lipid ROS levels. Furthermore, colony formation assays and Transwell assays showed that miR-612 could inhibit the proliferation and metastasis of HCC cells by promoting ferroptosis. We next confirmed that miR-612 influenced HCC cell ferroptosis by regulating HADHA. HADHA could upregulate the expression of key enzymes in the mevalonate (MVA) pathway. HADHA overexpression upregulated the expression of CoQ10 and decreased polyunsaturated fatty acid (PUFA) levels and lipid peroxide abundance. miR-612 also suppressed HCC cell proliferation and metastasis by enhancing RSL3- and lovastatin-induced ferroptosis in vivo. Conclusion: Overall, miR-612 promotes ferroptosis in HCC cells and affects HCC proliferation and metastasis by downregulating CoQ10 and increasing cellular PUFA levels and lipid peroxides via the HADHA-mediated MVA pathway.
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BACKGROUND: Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. METHODS: We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. RESULTS: GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. CONCLUSIONS: In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ⺠Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. âºDC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. âºCompared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Interleucina-7 , Glipicanas , Linhagem Celular Tumoral , Microambiente Tumoral , Quimiocina CCL19RESUMO
Metastasis is a landmark event for rapid postsurgical relapse and death of HCC patients. Although distinct genomic and transcriptomic profiling of HCC metastasis had been reported previously, the causal relationships of somatic mutants, mRNA levels and metastatic potentials were difficult to be established in clinic. Therefore, 11 human HCC cell lines and 7 monoclonal derivatives with definite metastatic potentials and tropisms were subjected to whole exome sequencing (WES) and whole transcriptome sequencing (WTS). TP53, MYO5A, ROS1 and ARID2 were the prominent mutants of metastatic drivers in HCC cells. During HCC clonal evaluation, TP53, MYO5A and ROS1 mutations occurred in the early stage, EXT2 and NIN in the late stage. NF1 mutant was unique in lung tropistic cell lines, RNF126 mutant in lymphatic tropistic ones. PER1, LMO2, GAS7, NR4A3 expression levels were positively associated with relapse-free survival (RFS) of HCC patients. The integrative analysis revealed 58 genes exhibited both somatic mutation and dysregulated mRNA levels in high metastatic cells. Altogether, metastatic drivers could accumulate gradually at different stages during HCC progression, some drivers might modulate HCC metastatic potentials and the others regulate metastatic tropisms.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Transcriptoma/genética , Proteínas Tirosina Quinases/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas/metabolismo , Genômica , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
[This corrects the article DOI: 10.7150/thno.46006.].
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As the first rate-limiting enzyme in fatty acid oxidation (FAO), CPT1 plays a significant role in metabolic adaptation in cancer pathogenesis. FAO provides an alternative energy supply for cancer cells and is required for cancer cell survival. Given the high proliferation rate of cancer cells, nucleotide synthesis gains prominence in rapidly proliferating cells. In the present study, we found that CPT1A is a determining factor for the abnormal activation of FAO in nasopharyngeal carcinoma (NPC) cells. CPT1A is highly expressed in NPC cells and biopsies. CPT1A dramatically affects the malignant phenotypes in NPC, including proliferation, anchorage-independent growth, and tumor formation ability in nude mice. Moreover, an increased level of CPT1A promotes core metabolic pathways to generate ATP, inducing equivalents and the main precursors for nucleotide biosynthesis. Knockdown of CPT1A markedly lowers the fraction of 13C-palmitate-derived carbons into pyrimidine. Periodic activation of CPT1A increases the content of nucleoside metabolic intermediates promoting cell cycle progression in NPC cells. Targeting CPT1A-mediated FAO hinders the cell cycle G1/S transition. Our work verified that CPT1A links FAO to cell cycle progression in NPC cellular proliferation, which supplements additional experimental evidence for developing a therapeutic mechanism based on manipulating lipid metabolism.
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Carnitina O-Palmitoiltransferase , Neoplasias Nasofaríngeas , Animais , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Proliferação de Células , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Camundongos , Camundongos Nus , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Nucleosídeos/metabolismo , Nucleotídeos/metabolismo , OxirreduçãoRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is inflammation-associated cancer with high incidence and poor prognosis. In the last decade, immunotherapy has become an important strategy for managing HCC. OBJECTIVE: This study aimed to establish an immune-related gene signature for predicting prognosis and immunotherapy response in HCC. METHODS: We identified immune-related differentially expressed genes (IRDEGs) based on The Cancer Genome Atlas (TCGA) database and the Immunology Database and Analysis Portal (ImmPort) database. The weighted gene co-expression network analysis (WGCNA) and Cox proportional hazard model were utilized to determine hub immune-related genes (IRGs). The TIDE tool and R package pRRophetic were used to assess the correlation between the immune-related gene signature and the clinical responses to immunotherapy and chemotherapy. RESULTS: By using WGCNA combined with Cox proportional hazard model, PRC1, TOP2A, TPX2, and ANLN were identified as hub IRGs. The prognostic value of the newly developed gene signature (IRGPI) was demonstrated in both the TCGA database and the Gene Expression Omnibus (GEO) database. The TIDE tool showed that the high- and low-IRGPI groups presented significantly different tumor immune microenvironment and immunotherapy responses. Furthermore, the high-IRGPI group also had significantly lower chemoresistance to cisplatin than the low-IRGPI group. CONCLUSION: The IRGPI is a tool for predicting prognosis as well as responsiveness to immunotherapy and chemotherapy in HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Cisplatino , Humanos , Imunoterapia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Microambiente Tumoral/genéticaRESUMO
In this study, batch experiments were conducted to investigate the immobilization of HMs (Cr and Pb) by DOM derived from biochar in the presence and absence of zero-valent iron (Fe) in nitrate and HMs co-contaminated groundwater. Both Cr and Pb were removed effectively in biochar-Fe aqueous systems, while only Pb could be mitigated in biochar systems. Excitation-emission spectrophotometry combined with parallel factor analysis (EEM-PARAFAC) revealed that DOM released from biochar mainly contained human-like and tryptophan-like substances. Moreover, the fluorescence of hemic-like components could be quenched differently by the complexation of HMs, which proved the different removal efficiencies of Cr and Pb in biochar aqueous phase. In biochar-Fe aqueous systems, Fe-C micro-electrolysis was formed in prior to the complexation of DOM-Fe hydroxides. Thus, the chemical reduction was the primary way to removal HMs in batch-Fe systems, which was corresponding with the less variation of DOM components when adding Cr and Pb into aqueous systems. Besides, the observed DOM components with higher aromaticity and humification after adding Cr and Pb, further indicated the complexation of DOM-HMs through the analysis of adsorption and fluorescence indices. These results will provide new insights into the HMs retention on biochar, particularly for the role of Fe on the complexation process.
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Ferro , Metais Pesados , Carvão Vegetal , Humanos , ChumboRESUMO
OBJECTIVE: To investigate the efficacy of an herbal formula of Bushen Jianpi ( BSJP) combined with sorafenib on hepatocellular carcinoma (HCC) in vitro and in vivo, and to study the underlying mechanisms of action. METHODS: BSJP, a mixture of 12 raw herbs, was extracted in 70% alcohol/30% water and freeze-dried into a powder. The in vitro effects of BSJP alone, sorafenib alone, and their combination on cell survival, apoptosis, and cell cycle distribution were evaluated in HCC cell lines HCCLM3, HepG2, and SMMC-7721. The expression of B-cell lymphoma-2 (Bcl-2), caspase-3, and caspase-9 in HCCLM3 cells was measured using Western blots after drug administration. The in vivo effects of BSJP and sorafenib were evaluated in a tumor surgical resection model using 4-week old male athymic BALB/c nude mice injected with HCCLM3 cells. Immunohistochemical analysis of tumor tissues was performed to evaluate the effects of BSJP alone, sorafenib alone, and their combination on the expression of caspase-3, caspase-9, and Bcl-2. RESULTS: BSJP decreased the survival rate of HCC cell lines, and the combination of BSJP and sorafenib further decreased the survival rate. BSJP significantly promoted cell apoptosis and blocked cell-cycle progression in HCCLM3, HepG2, and SMMC-7721 cells in a dose-dependent manner. Furthermore, the administration of BSJP and sorafenib inhibited the growth of HCCLM3 cell xenografts in nude mice, with no reduction in body weight. In vivo and in vitro experiments showed that BSJP combined with sorafenib could significantly decrease the expression of Bcl-2. CONCLUSION: Our findings suggest that the herbal formula of BSJP is a potential HCC antitumor agent.
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Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/fisiopatologia , Medicamentos de Ervas Chinesas/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/fisiopatologia , Sorafenibe/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimioterapia Combinada , Humanos , Masculino , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: S-adenosylmethionine decarboxylase proenzyme (AMD1) is a key enzyme involved in the synthesis of spermine (SPM) and spermidine (SPD), which are associated with multifarious cellular processes. It is also found to be an oncogene in multiple cancers and a potential target for tumor therapy. Nevertheless, the role AMD1 plays in hepatocellular carcinoma (HCC) is still unknown. METHODS: HCC samples were applied to detect AMD1 expression and evaluate its associations with clinicopathological features and prognosis. Subcutaneous and orthotopic tumor mouse models were constructed to analyze the proliferation and metastasis of HCC cells after AMD1 knockdown or overexpression. Drug sensitive and tumor sphere assay were performed to investigate the effect of AMD1 on HCC cells stemness. Real-time quantitative PCR (qRT-PCR), western blot, immunohistochemical (IHC) and m6A-RNA immunoprecipitation (Me-RIP) sequencing/qPCR were applied to explore the potential mechanisms of AMD1 in HCC. Furthermore, immunofluorescence, co-IP (Co-IP) assays, and mass spectrometric (MS) analyses were performed to verify the proteins interacting with AMD1. RESULTS: AMD1 was enriched in human HCC tissues and suggested a poor prognosis. High AMD1 level could promote SRY-box transcription factor 2 (SOX2), Kruppel like factor 4 (KLF4), and NANOG expression of HCC cells through obesity-associated protein (FTO)-mediated mRNA demethylation. Mechanistically, high AMD1 expression increased the levels of SPD in HCC cells, which could modify the scaffold protein, Ras GTPase-activating-like protein 1 (IQGAP1) and enhance the interaction between IQGAP1 and FTO. This interaction could enhance the phosphorylation and decrease the ubiquitination of FTO. CONCLUSIONS: AMD1 could stabilize the interaction of IQGAP1 with FTO, which then promotes FTO expression and increases HCC stemness. AMD1 shows prospects as a prognostic predictor and a therapeutic target for HCC.
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Adenosilmetionina Descarboxilase/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Carcinoma Hepatocelular/genética , Desmetilação , Neoplasias Hepáticas/genética , RNA Mensageiro/metabolismo , Adenosilmetionina Descarboxilase/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/genética , Modelos Animais de Doenças , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Mensageiro/genética , Células-Tronco/metabolismo , Regulação para Cima/genéticaRESUMO
BACKGROUND: lncRNAs-miRNAs-mRNAs networks play an important role in Gastric adenocarcinoma (GA). Identification of these networks provide new insight into the role of these RNAs in gastric cancer. OBJECTIVES: Biological information databases were screened to characterize and examine the regulatory networks and to further investigate the potential prognostic relationship this regulation has in GA. METHODS: By mining The Cancer Genome Atlas (TCGA) database, we gathered information on GA-related lncRNAs, miRNAs, and mRNAs. We identified differentially expressed (DE) lncRNAs, miRNAs, and mRNAs using R software. The lncRNA-miRNA-mRNA interaction network was constructed and subsequent survival examination was performed. Representative genes were selected out using The Biological Networks Gene Ontology plug-in tool on Cytoscape. Additional analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms were used to screen representative genes for functional enrichment. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to identify the expression of five candidate differential expressed RNAs. RESULTS: Information of samples from 375 cases of gastric cancer and 32 healthy cases (normal tissues) were downloaded from the TCGA database. A total of 1632 DE-mRNAs, 1008 DE-lncRNAs and 104 DE-miRNAs were identified and screened. Among them, 65 DE-lncRNAs, 10 DE-miRNAs, and 10 DE-mRNAs form lncRNAs-miRNAs-mRNAs regulatory network. Additionally, 10 lncRNAs and 2 mRNAs were associated with the prognosis of GA. Multivariable COX analysis revealed that AC018781.1 and VCAN-AS1 were independent risk factors for GA. GO functional enrichment analysis found DE-mRNA was significantly enriched TERM (P < 0.05). The KEGG signal regulatory network analysis found 11 significantly enrichment networks, the most prevailing was for the AGE-RAGE signaling pathway associated with Diabetic complications. Results of RT-qPCR was consistent with the in silico results. CONCLUSIONS: The results of the present study represent a view of GA from a analysis of lncRNA, miRNA and mRNA. The network of lncRNA-miRNA-mRNA interactions revealed here may potentially further experimental studies and may help biomarker development for GA.
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Adenocarcinoma/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Neoplasias Gástricas/genética , Adenocarcinoma/epidemiologia , Adenocarcinoma/patologia , Biomarcadores Tumorais/genética , Biologia Computacional , Bases de Dados Genéticas , Epistasia Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Ontologia Genética , Redes Reguladoras de Genes/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Prognóstico , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/patologiaRESUMO
p18 is a key negative regulator of cell cycle progression and mediates cell cycle arrest at the G1/S phase. Ubiquitination is the prime mechanism in regulating p18 protein abundance. However, so far no post- translational regulator, especially DUBs, has been identified to regulate the protein stability of p18. In this paper, we identified CYLD as a deubiquitinase of p18, which binds to and removes the K48-linked polyubiquitylation chains conjugated onto p18, thus stabilizing the p18 protein. Loss of CYLD causes the degradation of p18 and induces the G1/S transition. Epstein-Barr virus (EBV), is the human oncovirus etiologically linked to nasopharyngeal carcinoma (NPC). Here we found that EBV drives a replication passive environment by deregulating the CYLD-p18 axis. Functionally, CYLD inhibits cell proliferation and tumorigenesis through p18 in vivo. Restoring CYLD prevents EBV induced viral replication and tumor growth. Collectively, our results identify CYLD directly stabilizes p18 to regulate the cellular G1/S transition. The reconstitution of CYLD-p18 axis could be a promising approach for EBV-positive cancer therapy.
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Generating oxidative stress is a critical mechanism by which host cells defend against infection by pathogenic microorganisms. Radiation resistance is a critical problem in radiotherapy against cancer. Epstein-Barr virus (EBV) is a cancer-causing virus and its reactivation plays an important role in the development of EBV-related tumors. This study aimed to explore the inner relationship and regulatory mechanism among oxidative stress, EBV reactivation, and radioresistance and to identify new molecular subtyping models and treatment strategies to improve the therapeutic effects of radiotherapy. Methods: ROS, NADP+/NADPH, and GSSG/GSH were detected to evaluate the oxidative stress of cells. 8-OHdG is a reliable oxidative stress marker to evaluate the oxidative stress in patients. Its concentration in serum was detected using an ELISA method and in biopsies was detected using IHC. qPCR array was performed to evaluate the expression of essential oxidative stress genes. qPCR, Western blot, and IHC were used to measure the level of EBV reactivation in vitro and in vivo. A Rta-IgG ELISA kit and EBV DNA detection kit were used to analyze the reactivation of EBV in serum from NPC patients. NPC tumor tissue microarrays was used to investigate the prognostic role of oxidative stress and EBV reactivation. Radiation resistance was evaluated by a colony formation assay. Xenografts were treated with NAC, radiation, or a combination of NAC and radiation. EBV DNA load of tumor tissue was evaluated using an EBV DNA detection kit. Oxidative stress, EBV reactivation, and the apoptosis rate in tumor tissues were detected by using 8-OHdG, EAD, and TUNEL assays, respectively. Results: We found that EBV can induce high oxidative stress, which promotes its reactivation and thus leads to radioresistance. Basically, EBV caused NPC cells to undergo a process of 'Redox Resetting' to acquire a new redox status with higher levels of ROS accumulation and stronger antioxidant systems by increasing the expression of the ROS-producing enzyme, NOX2, and the cellular master antioxidant regulator, Nrf2. Also, EBV encoded driving protein LMP1 promotes EBV reactivation through production of ROS. Furthermore, high oxidative stress and EBV reactivation were positively associated with poor overall survival of patients following radiation therapy and were significant related to NPC patients' recurrence and clinical stage. By decreasing oxidative stress using an FDA approved antioxidant drug, NAC, sensitivity of tumors to radiation was increased. Additionally, 8-OHdG and EBV DNA could be dual prognostic markers for NPC patients. Conclusions: Oxidative stress mediates EBV reactivation and leads to radioresistance. Targeting oxidative stress can provide therapeutic benefits to cancer patients with radiation resistance. Clinically, we, for the first time, generated a molecular subtyping model for NPC relying on 8-OHdG and EBV DNA level. These dual markers could identify patients who are at a high risk of poor outcomes but who might benefit from the sequential therapy of reactive oxygen blockade followed by radiation therapy, which provides novel perspectives for the precise treatment of NPC.
Assuntos
Quimiorradioterapia/métodos , Infecções por Vírus Epstein-Barr/terapia , Sequestradores de Radicais Livres/administração & dosagem , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/terapia , Acetilcisteína/administração & dosagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biópsia , Linhagem Celular Tumoral , DNA Viral/sangue , Infecções por Vírus Epstein-Barr/sangue , Infecções por Vírus Epstein-Barr/mortalidade , Infecções por Vírus Epstein-Barr/virologia , Feminino , Seguimentos , Dissulfeto de Glutationa/sangue , Dissulfeto de Glutationa/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Herpesvirus Humano 4/patogenicidade , Humanos , Infecção Latente/sangue , Infecção Latente/patologia , Infecção Latente/terapia , Infecção Latente/virologia , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/sangue , Carcinoma Nasofaríngeo/mortalidade , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/sangue , Neoplasias Nasofaríngeas/mortalidade , Neoplasias Nasofaríngeas/virologia , Nasofaringe/patologia , Nasofaringe/virologia , Estresse Oxidativo/efeitos dos fármacos , Seleção de Pacientes , Prognóstico , Intervalo Livre de Progressão , Tolerância a Radiação/efeitos dos fármacos , Espécies Reativas de Oxigênio , Carga Viral , Proteínas da Matriz Viral/metabolismo , Ativação Viral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Latent membrane protein 1 (LMP1) is a major Epstein-Barr virus (EBV)-encoded oncoprotein involved in latency infection that regulates mitochondrial functions to facilitate cell survival. Recently, mitochondrial fission has been demonstrated as a crucial mechanism in oncovirus-mediated carcinogenesis. Mitochondrial dynamin-related protein 1 (Drp1)-mediated mitochondrial fission has an impact on the chemoresistance of cancers. However, the mechanism by which oncogenic stress promotes mitochondrial fission, potentially contributing to tumorigenesis, is not entirely understood. The role of Drp1 in the oncogenesis and prognosis of EBV-LMP1-positive nasopharyngeal carcinoma (NPC) was determined in our study. We show that EBV-LMP1 exhibits a new function in remodeling mitochondrial morphology by activating Drp1. A high level of p-Drp1 (Ser616) or a low level of p-Drp1 (Ser637) correlates with poor overall survival and disease-free survival. Furthermore, the protein level of p-Drp1 (Ser616) is related to the clinical stage (TNM stage) of NPC. Targeting Drp1 impairs mitochondrial function and induces cell death in LMP1-positive NPC cells. In addition, EBV-LMP1 regulates Drp1 through two oncogenic signaling axes, AMPK and cyclin B1/Cdk1, which promote cell survival and cisplatin resistance in NPC. Our findings provide novel insight into the role of EBV-LMP1-driven mitochondrial fission in regulating Drp1 phosphorylation at serine 616 and serine 637. Disruption of Drp1 could be a promising therapeutic strategy for LMP1-positive NPC.
Assuntos
Dinaminas/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Linhagem Celular Tumoral , Dinaminas/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Feminino , Herpesvirus Humano 4/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/genética , Mitocôndrias/patologia , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patologia , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , Proteínas de Neoplasias/genética , Proteínas da Matriz Viral/genéticaRESUMO
An amendment to this paper has been published and can be accessed via the original article.
RESUMO
OBJECTIVE: Epstein-Barr virus (EBV) is a well-recognized oncogenic virus that can induce host cell metabolic reprogramming and tumorigenesis by targeting vital metabolic enzymes or regulators. This study aims to explore the role of wild-type isocitrate dehydrogenase 2 (IDH2) in metabolic reprogramming and tumorigenesis induced by EBV-encoded latent membrane protein 1 (LMP1). METHODS: Mechanistic dissection of wild-type IDH2 in EBV-LMP1-induced tumorigenesis was investigated using western blotting, real-time polymerase chain reaction (PCR), immunochemistry, chromatin immunoprecipitation (ChIP), and luciferase assay. The role of wild-type IDH2 was examined by cell viability assays/Sytox Green staining in vitro and xenograft assays in vivo. RESULTS: IDH2 over-expression is a prognostic indicator of poorer disease-free survival for patients with head and neck squamous cell carcinoma (HNSCC). IDH2 expression is also upregulated in nasopharyngeal carcinoma (NPC, a subtype of HNSCC) tissues, which is positively correlated with EBV-LMP1 expression. EBV-LMP1 contributes to NPC cell viability and xenograft tumor growth mediated through wild-type IDH2. IDH2-dependent changes in intracellular α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG) contribute to EBV-LMP1-induced tumorigenesis in vitro and in vivo. Elevated serum 2-HG level is associated with high EBV DNA and viral capsid antigen-immunoglobulin A (VCA-IgA) levels in patients with NPC. A significantly positive correlation exists between serum 2-HG level and regional lymph node metastases of NPC. EBV-LMP1 enhances the binding of c-Myc with the IDH2 promoter and transcriptionally activates wild-type IDH2 through c-Myc. Targeting IDH2 decreased intracellular 2-HG levels and survival of EBV-LMP1-positive tumor cells in vitro and in vivo. CONCLUSIONS: Our results demonstrate that the EBV-LMP1/c-Myc/IDH2WT signaling axis is critical for EBV-dependent metabolic changes and tumorigenesis, which may provide new insights into EBV-related cancer diagnosis and therapy.
Assuntos
Carcinogênese/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Isocitrato Desidrogenase/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular , Linhagem Celular Tumoral , China , Bases de Dados Genéticas , Feminino , Herpesvirus Humano 4/patogenicidade , Humanos , Isocitrato Desidrogenase/fisiologia , Masculino , Camundongos , Camundongos Nus , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/virologia , Regiões Promotoras Genéticas/genética , Transdução de Sinais , Proteínas da Matriz Viral/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epithelial-mesenchymal transition (EMT), a pivotal event during cancer progression such as relapse and metastasis, is positively correlated with the stemness potency of tumor cells. Our previous study showed that miR-296-5p attenuated EMT program of hepatocellular carcinoma cells (HCC) through NRG1/ERBB2/ERBB3 signaling. In the present study, we uncovered that miR-296-5p was able to inhibit the stemness potency of HCC by decreasing the number and size of tumorspheres, downregulating the expression of CSC biomarkers and hampering the ability of tumorigenesis in NOD/SCID mice. Brahma-related gene-1 (Brg1), as the target protein of miR-296-5p detected by bioinformatics methods, activates a series of downstream cascades through directly binding to Sall4 promoter and enhancing Sall4 transcription. Importantly, the higher expressions of Brg1 and Sall4 in tumor tissues of HCC patients suggest poorer prognoses after surgical extraction. In conclusion, miR-296-5p exerts an inhibitory effect on stemness potency of HCC cells via Brg1/Sall4 axis.